Abstract Expansion Microscopy (ExM) enhances biological imaging to the nanoscale level by enlarging biological specimens, increasing the separation between biomolecules while maintaining their spatial arrangement. By integrating ExM with traditional microscopy, super-resolution volumetric images can be captured efficiently without compromising tissue integrity. Accordingly, fine molecular structures previously indiscernible by means of standard fluorescence microscopy can be visualized. However, one of the major challenges with ExM is that the samples are embedded in the polymerized gel, which limits the use of high numerical aperture objective lenses due to their short working distances. To address this issue, we have developed an approach using carboxymethyl tamarind kernel gum (CMTKG) cross-linked with sodium acrylate to create a superabsorbent hydrogel suitable for ExM. This technique facilitates removal of excess polymers from the expanded sample, resulting in sample exposure to the objective lens. Here, we demonstrate that it is possible to achieve approximately 7X expansion of Drosophila melanogaster brains, free from surrounding hydrogel, in a process we have named Naked-ExM. Followed by iterative expansion, the centimeter-sized fly brain is obtained and the ~15 um sized synapses are measured at 15X expansion.
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